Plasma extracellular vesicles bearing PD-L1, CD40, CD40L or TNF-RII are significantly reduced after treatment of AIDS-NHL

Emerging evidence shows that tumor cells secrete extracellular vesicles (EVs) that carry bioactive cell surface markers, such as programmed death-ligand 1 (PD-L1), which can modulate immune responses and inhibit anti-tumor responses, potentially playing a role in lymphomagenesis and in promoting the growth of these cancers. In this study, we investigated the role of EVs expressing cell surface molecules associated with B cell activation and immune regulation. We measured levels of EVs derived from plasma from 57 subjects with AIDS-related non-Hodgkin lymphoma (AIDS-NHL) enrolled in the AIDS Malignancies Consortium (AMC) 034 clinical trial at baseline and post-treatment with rituximab plus concurrent infusional EPOCH chemotherapy. We found that plasma levels of EVs expressing PD-L1, CD40, CD40L or TNF-RII were significantly reduced after cancer treatment. AIDS-NHL patients with the diffuse large B cell lymphoma (DLBCL) tumor subtype had decreased plasma levels of EVs bearing PD-L1, compared to those with Burkitt’s lymphoma. CD40, CD40L and TNF-RII-expressing EVs showed a significant positive correlation with plasma levels of IL-10, CXCL13, sCD25, sTNF-RII and IL-18. Our results suggest that patients with AIDS-NHL have higher levels of EVs expressing PD-L1, CD40, CD40L or TNF-RII in circulation before cancer treatment and that levels of these EVs are associated with levels of biomarkers of microbial translocation and inflammation.

. Characterization of extracellular vesicles isolated from plasma of AMC-034 trial participants at pre-treatment (baseline) and post-treatment. (A) Western blot analysis of EVs demonstrating the presence of exosome-specific markers, such as the tetraspanin CD9, TSG101, and HSP70, and (B) a non-exosomal maker, Calnexin (endoplasmic reticulum marker). 20 µg was used from each sample. Results are from plasma-derived EVs of matched pre-treatment (pre-RX or baseline) (N = 3) and post-treatment (post-Rx) (N = 3) plasma samples of AMC-034 trial subjects, including protein from the lysate of the Raji cell line (20 µg). The exposure time for the HSP70 blot was 90 s. For the TSG101 blot, boxed lines delineate that AMC pre-and post-Rx sample 3 was run in a separate gel, and blots were exposed for 30 s each. The exposure time for the CD9 blot was 120 s. Full blots are shown for Calnexin, where AMC pre-and post-Rx sample 3 was run in a separate gel along with the cell lysate from the Raji cell line. Each blot was exposed for 30 s. www.nature.com/scientificreports/ marker and it was only detected in protein from lysate of Raji cells, a Burkitt's lymphoma cell line (Fig. 1B), but was not detected in EVs isolated from the plasma of AIDS-NHL patients.

AIDS-NHL patients showed decreased plasma levels of EVs bearing molecules associated with immune activation following cancer treatment.
We then evaluated whether EVs expressed cell surface markers on their surfaces that are important in immune regulatory responses and that play a role in lymphomagenesis. Thus, we measured plasma levels of EVs expressing molecules important for B and T cell signaling (CD40 and CD40L), molecules with immune modulatory activities (PD-L1, B7 immune checkpoint molecule B7-H3 [CD276], and ICAM-1 [CD54)]), TNF-RII, the receptor for TNFα and an indicator of clinical response to treatment and survival in AIDS-NHL 16,17 ; IL-6Rα, which is part of the IL-6 receptor, and a molecule involved in the regulation of cell death, Fas Ligand (FasL). We measured the expression of these cell surface markers on EVs (PD-L1, CD40, CD40L, B7-H3, TNF-RII, IL-6Rα, ICAM-1 or FasL) using a multiplexed bead beased immunometric assay (Luminex). AIDS-NHL patients showed decreased plasma levels of EVs bearing PD-L1 (p = 0.006), CD40 (p = 0.003), CD40L (p < 0.001) or TNF-RII (p = 0.015) after cancer treatment, compared to baseline plasma levels (Wilcoxon signed rank test) ( Fig. 2A-D). The data presented in Fig. 2  Plasma-derived EVs expressing PD-L1 were significantly lower in AIDS-NHL patients with the DLBCL tumor subtype. We then evaluated any associations between blood circulating EVs expressing the different immune cell surface markers (PD-L1, CD40, CD40L, B7-H3, TNF-RII, IL-6Rα, ICAM-1 or FasL) and tumor subtype of AIDS-NHL patients. AIDS-NHL patients who had the DLBCL tumor subtype showed significantly reduced plasma levels of PD-L1-expressing EVs when compared to patients with Burkitt's lymphoma tumor subtype (p = 0.037, Wilcoxon two-sample test) (Fig. 3). We further compared EVs according to IPI score for AIDS-NHL patients with DLBCL tumor subtype, and found that the group with IPI scores between 2-3 had significantly higher EVs bearing PD-L1, CD40, CD40L or TNF-RII compared to the group with an IPI score between 0 and 1 (Fig. 4).

Discussion
Extracellular vesicles released from cancer cells can function as messengers to regulate other cells in the tumor microenvironment, and contribute to tumor growth and tumor progression 35 . PD-L1 is highly expressed in tumor cells and can be secreted in EVs to alter the immune system response in the tumor microenvironment 25 .
In this study, we measured plasma concentrations of PD-L1-expressing EVs and compared those with clinical and pathologic features of patients with AIDS-NHL who were taking part in the AMC-034 trial, such as HIV viral load, CD4 + T cell count, tumor subtype, response to cancer treatment, patient outcome measures, and evaluated associations with plasma levels of molecules important for AIDS-NHL risk 16,17 . We found that plasma levels of PD-L1-expressing EVs were significantly higher in AIDS-NHL patients prior to the initiation of rituximab and EPOCH chemotherapy. A significant correlation between plasma levels of EVs expressing PD-L1 and plasma levels of IL-10 was observed at baseline. It is important to note that DLBCL tumor subtype had overall reduced plasma levels of PD-L1 + EVs compared to the Burkitt's NHL subgroup, raising the possibility that the positive and significant association between PD-L1 + EVs and plasma levels of IL-10 is due to tumor type. Although this is difficult to acertain, since there were only seven patients with Burkitt's NHL, we did see that the correlation was stronger in the DLBCL subset compared to both tumor types combined. These results suggest that plasma levels of IL-10 at baseline may act as a negative immunomodulatory factor to inhibit immune responses associated with circulation of PD-L1 + EVs. We previously showed that pre-treatment levels of IL-10 were associated with response to therapy, as well as overall survival, in AIDS-NHL 16 . Thus, plasma levels of PD-L1-expressing EVs and IL-10 hold promise as effective prognostic biomarkers in AIDS-NHL.
We then examined other molecules important for B and T cell signaling, such as CD40, CD40L, and B7-H3 (CD276), which have co-stimulatory/co-inhibitory immunoregulatory functions 36 , and are highly expressed in different types of cancers 37 . We also evaluated the expression of TNF-RII on EVs, whose expression has been linked to tolerogenic immune reactions, including a subset of T-regulatory cells 38 and IL-10-producing B cells 39 ; and EVs expressing IL-6Rα, which is of high interest since our previous work has shown that IL-6 has prognostic value as an indicator of subsequent response to AIDS-NHL treatment and survival 16 . Moreover, we also quantified plasma levels of FasL-expressing EVs. FasL can be expressed in both membrane bound and soluble forms and has been shown to be secreted in vesicles from activated T cells to induce Fas-mediated apoptosis 40 .
We found that EVs bearing CD40 correlated with plasma levels of IL-10, CXCL13, sCD25, sTNF-RII, IL-18, and sCD163, while TNF-RII-expressing EVs correlated with IL-6, IL-10, CXCL13, sCD25, sTNF-RII, IL-18, BAFF, sCD14, sCD44, and CXCL10 at baseline. IL-6Rα-expressing EVs correlated with several plasma biomarkers, such as IL-10, CXCL13, sCD25, sTNF-RII, IL-18, sCD163, BAFF, and CXCL10 at baseline. Thus, we have found that molecules important for B cell signaling and that promote B cell survival are enriched in blood circulating EVs at baseline in AIDS-NHL patients, and that these plasma-derived EVs correlate with biomarkers lymphocytes. Moreover, we did not find significant differences in plasma levels of EVs bearing FasL at baseline and post-treatment, but we did find correlations between FasL-expressing EVs and plasma levels of biomarkers for AIDS-NHL risk (sCD44, CXCL10, and TNF-α) at baseline, suggesting that they can mediate tumor immune responses and/or tumor immune escape. Morever, the expression of these different immune cell surface molecules on plasma-derived EVs can mediate early events in lymphomagenesis and play a vital role in cancer metastasis, further interacting with different immune cells in the tumor microenvironment and modulating immune responses. EVs expressing PD-L1 can modulate T cell responses, promoting the growth of tumor cells and/or inhibiting T cells in distant draining lymph nodes 41 .
These results suggest that EVs may serve as potential biomarkers for prospective immunotherapy-focused clinical trials for AIDS-NHL prior to, and after, initiating therapy. Further studies will include the proteomic profiling of plasma-derived EVs from AIDS-NHL patients in order to define other immunoregulatory molecules associated with immune activation and/or dysfunction, as well as the cytokine profiling of EVs. Extracellular vesicle cargos are heterogeneous and mirror the landscape of EV producing cells. Therefore, it will be important to profile the repertoire of EV proteomes, which can be subjected to imaging mass cytometry to examine EVs in the tumor microenvironment.
Various studies have shown that HIV-1-infected cells secrete exosomes that carry viral RNA and proteins and that can be transported to other cells in the microenvironment [42][43][44][45][46] . These EVs have the potential to have myriad effects on the pathogenesis of AIDS-NHL and disease progression. Thus, it will be important to elucidate the molecular mechanisms mediated by plasma-derived EVs to determine if EVs bearing PD-L1 and/or other B7-molecules have immunosuppressive properties that may inhibit T cell activation and/or anti-tumor responses.
Circulating tumor-derived EVs are being examined for the early detection of a variety of cancers, which have the potential to discover new chemopreventive strategies and/or anti-cancer drug therapies. Our study supports that EVs are associated with biomarkers contributing to chronic B cell activation, macrophage activation, and microbial translocation, all factors that are associated with AIDS-NHL risk and pathogenesis. These findings have relevance to HIV-infected individuals with high levels of tumor cells secreting EVs in blood circulation while undergoing treatment with anti-B-cell therapy drugs, including rituximab and chemotherapy agents.  . Plasma was collected before the initiation of therapy at the end of the first cycle (within a week or less of treatment), and at 6 months and one year following the completion of treatment. were differentially centrifuged at 2000×g for 20 min at room temperature to remove cell debris. The supernatant of clarified plasma was then transferred to a new tube and centrifuged at 10,000×g for 20 min at room temperature to further remove any remaining debris. The supernatant containing the clarified plasma was then transferred to a new tube with 0.5 ml volume of 1× PBS and mixed thoroughly, and 0.2 ml volume of the exosome precipitation reagent (from plasma) was added to the sample (Total Exosome Isolation (Plasma) Kit, Invitrogen, Cat No:4484450). The plasma/reagent solution was mixed by vortexing until the solution was homogenous and then the samples were incubated at room temperature for 10 min. After the incubation period, samples were centrifuged at 10,000×g for 5 min at room temperature to pellet exosomes. The supernatant was retrieved by pipet and discarded. The EV pellet was centrifuged for an additional 30 s at 10,000×g to collect any residual reagent. The residual supernatant was carefully discarded with a pipet. Then, 300 μl of 1× PBS was added to the pellet, resuspended with a pipet, and then lightly vortexed. Isolated EVs were stored at 4 °C or kept at − 20 °C until ready for downstream analysis, such as for Luminex multiplex immunometric assay and western blot analysis. www.nature.com/scientificreports/ Cat#28283-1-AP) at 1:2000. All primary antibodies were diluted in 1× TBS with 0.2% Tween 20, and incubated overnight at 4 °C with gentle shaking. The next day, the blot was rinsed 3-5 times with 1× TBS-0.1% Tween 20 for 5 min each wash over a platform shaker, and then incubated with appropriate secondary antibodies: IRDye 800CW goat anti-mouse secondary antibody (1:10,000) (for detection of CD9 and Calnexin), IRDye 680RD goat anti-rabbit secondary antibody (1:10,000) (for detection of TSG101) or IRDye 800CW donkey anti-rabbit secondary antibody (1:10,000) (for detection of HSP70). Secondary antibodies were diluted in 1× TBS with 0.2% Tween 20 and 0.02% SDS for 1 h at room temperature with gentle shaking. The blot was then washed 3 times with 1× TBS-0.1% Tween 20 for 5 min each wash while shaking vigorously over a platform shaker at room temperature, and then finally rinsed in 1× TBS to remove residual Tween 20. Blots were then immediately scanned at 700 nm or 800 nm using a ChemiDoc Touch Imaging System (UCLA AIDS Institute) and images were collected.

Extracellular vesicle protein quantification and western blot analysis.
Quantification of plasma-derived EVs expressing PD-L1 and other immunoregulatory molecules by Luminex multiplex immunometric assay. EVs  In some instances, extrapolated values were utilized, which are values that are based on a fluorescence signal that is above the background level of detectable fluorescence for the Luminex analyzer, but below the lowest value of the standard curve. For quality control, pre-treatment and post-treatment exosome samples were equally distributed across reaction plates, and duplicates were included across the reaction plates to calculate coefficients of variation. Laboratory personnel were blinded to the pre-and post-treatment status of samples.
To determine associations between EVs and biomarkers previously identified for AIDS-NHL risk, we used data previously acquired and reported in Ref. 17 . Briefly, plasma levels of EndoCab IgM (Hycult Biotech, Uden, The Netherlands) were determined by ELISA, according to the manufacturers' instructions. Plasma levels of all other biomarkers (sCD14, LBP, FABP2, IL-18, CCL2/MCP-1, sCD163, IP-10/CXCL10, TARC/CCL17, TNF-α, BAFF/BLyS, sTNF-RII, sCD44, and sIL2Rα/sCD25) were determined using the Luminex multiplex assay platform with custom-made panels, as previously described 17 . Statistical analysis. Changes in plasma-derived EVs bearing PD-L1 were compared pre-to post-cancer treatment using paired nonparametric Wilcoxon sign-rank tests. In addition, pre-treatment (baseline) biomarkers were compared according to tumor type using the nonparametric Wilcoxon rank sum test. The relationships between PD-L1, CD40, CD40L, TNF-RII, IL-6Rα, B7-H3, ICAM-1 or FasL-expressing EVs and plasma levels of cytokines, chemokines, and prognostic biomarkers of AIDS-NHL were assessed using Spearman's rank correlation coefficient. p values were not adjusted for multiple comparisons. In all cases, a two-tailed value of p < 0.05 was considered statistically significant.
Informed consent and regulatory approval. The study was reviewed and approved by the Cancer Evaluation Therapy Program of the National Cancer Institute, and by the institutional review board at each participating institution. All patients provided written informed consent in accordance with the Declaration of Helsinki.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request and AMC approval. Methods were performed in accordance to the relevant guidelines and regulation.